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R Oliva

Publications and source records attributed to R Oliva.

At least 91 records · Page 5Linked to original sources
PubMed1993

Direct sequencing of the human protamine P1 gene and application in forensic medicine.

Protamines are among the most variable nuclear proteins known in eukaryotes. In order to learn more about their evolution and function in humans and to explore the possibility of potential applications in forensic medicine we have developed a rapid method to amplify and directly sequence the protamine P1 gene simultaneously in many different samples. The method takes only 3.5 h from genomic DNA to the sequencing reactions. Despite the high variability of these genes only one polymorphic site was detected at the coding region level in different individuals. This polymorphic variation does not create a change in the amino-acid sequence of the protamine. Because all the protamine genes sequenced from different species are markedly different among them as well as to the human sequence, amplification and direct sequencing of this gene can be used to unequivocally identify the human or animal origin of biological specimens. Furthermore, the single polymorphic site detected in the human P1 gene could be useful in conjunction with other markers in identification studies in humans.

Amino Acid Sequence↗
PubMed1992

Improved direct sequencing of Alzheimer's amyloid precursor protein (APP) exons 16 and 17.

Direct sequencing of exon 17 of the amyloid precursor protein (APP) gene led to the identification of 3 different types of APP717 pathogenic mutations associated with familial Alzheimer's disease (FAD). The low frequency of these mutations results in having to screen many samples in order to identify new families affected by them, which is laborious and time consuming. Thus, in order to help the identification of these mutations in additional countries and to search for new mutations in APP, perhaps in other exons also causing FAD, we have optimized the procedure and reduced the time necessary for sample preparation from 11 h to 3 1/2 h.

Alzheimer Disease↗
PubMed1992

Long-distance restriction mapping of the proximal long arm of human chromosome 21 with Not I linking clones.

Human chromosome 21 is the smallest of the 22 autosomes and 2 sex chromosomes. Hybridization of the human repetitive sequence Alu to pulsed-field gel-fractionated Not I-digested genomic DNA from a human-mouse hybrid cell line containing chromosome 21 as the sole human component identified chromosome 21 Not I restriction fragments. A Not I restriction map of regions of the chromosome was constructed, by identifying neighboring Alu bands with Not I linking clones. This approach simplifies the task of physical mapping and avoids ambiguities in Not I fragment assignments that arise from gel-to-gel mobility variations. A contiguous map was constructed with six Not I linking clones that covers at least the proximal one-third of the long arm of chromosome 21 and spans 20 megabases. A more detailed restriction map revealed 11 likely CpG islands in this region and localized 11 additional DNA markers.

Chromosomes, Human, Pair 21↗
PubMed1991

Expression and processing of the rooster protamine mRNA.

In situ hybridization in immature and mature testis sections shows that the rooster protamine mRNA is transcribed in the post-meiotic stages of spermatogenesis. Two distinct populations of rooster protamine mRNA are expressed as determined by Northern blot analysis. Since there are two copies of the chicken protamine gene per haploid genome, the question was raised of whether the two mRNA populations correspond to the two different genes or was a result of differential mRNA processing. The fact that the two genes differ only in one nucleotide (one extra A in the polyadenylation signal in the second locus) and that random sequencing of several cDNA clones has revealed only one poly-A tail addition site favors the hypothesis that the differences are due to mRNA processing. This is supported by 3' S1 mapping which shows a single poly-A tail addition site, which in turn suggests that the heterogeneity is due to differences in the length of the poly-A tail. The latter is confirmed by RNAse H digestion of mRNA-Oligo-dT hybrids which shows that the two mRNA populations (470 +/- 20 nucleotide (nt) and 430 +/- 20 nt respectively) are converted to a single population of 345 +/- 15 nt in good accordance with the poly-A tail site determined by S1 mapping (347 nt). Thus one species has a poly-A tail of 145 nt appearing in round spermatids and the second, a shorter tail of 105 nt present at the later stages of elongated spermatids.

Amino Acid Sequence↗
PubMed1990

Histone hyperacetylation can induce unfolding of the nucleosome core particle.

A direct correlation exists between the level of histone H4 hyperacetylation induced by sodium butyrate and the extent to which nucleosomes lose their compact shape and become elongated (62.0% of the particles have a length/width ratio over 1.6; overall mean in the length/width ratio = 1.83 +/- 0.48) when bound to electron microscope specimen grids at low ionic strength (1mM EDTA, 10mM Tris, pH 8.0). A marked proportion of elongated core particles is also observed in the naturally occurring hyperacetylated chicken testis chromatin undergoing spermatogenesis when analyzed at low ionic strength (36.8% of the particles have a length/width ratio over 1.6). Core particles of elongated shape (length/width ratio over 1.6) generated under low ionic strength conditions are absent in the hypoacetylated chicken erythrocyte chromatin and represent only 2.3% of the untreated Hela S3 cell core particles containing a low proportion of hyperacetylated histones. The marked differences between control and hyperacetylated core particles are absent if the particles are bound to the carbon support film in the presence of 0.2 M NaCl, 6mM MgCl2 and 10mM Tris pH 8.0, conditions known to stabilize nucleosomes. A survey of the published work on histone hyperacetylation together with the present results indicate that histone hyperacetylation does not produce any marked disruption of the core particle 'per se', but that it decreases intranucleosomal stabilizing forces as judged by the lowered stability of the hyperacetylated core particle under conditions of shearing stress such as cationic competition by the carbon support film of the EM grid for DNA binding.

Acetylation↗
PubMed1990

Vertebrate protamine gene evolution I. Sequence alignments and gene structure.

The availability of the amino acid sequence for nine different mammalian P1 family protamines and the revised amino acid sequence of the chicken protamine galline (Oliva and Dixon 1989) reveals a much close relationship between mammalian and avian protamines than was previously thought (Nakano et al. 1976). Dot matrix analysis of all protamine genes for which genomic DNA or cDNA sequence is available reveals both marked sequence similarities in the mammalian protamine gene family and internal repeated sequences in the chicken protamine gene. The detailed alignments of the cis-acting regulatory DNA sequences shows several consensus sequence patterns, particularly the conservation of a cAMP response element (CRE) in all the protamine genes and of the regions flanking the TATA box, CAP site, N-terminal coding region, and polyadenylation signal. In addition we have found a high frequency of the CA dinucleotide immediately adjacent to the CRE element of both the protamine genes and the testis transition proteins, a feature not present in other genes, which suggests the existence of an extended CRE motif involved in the coordinate expression of protamine and transition protein genes during spermatogenesis. Overall these findings suggest the existence of an avian-mammalian P1 protamine gene line and are discussed in the context of different hypotheses for protamine gene evolution and regulation.

Amino Acid Sequence↗
PubMed1989

Quail (Coturnix japonica) protamine, full-length cDNA sequence, and the function and evolution of vertebrate protamines.

Using the chicken protamine gene as a probe, we have isolated and sequenced several positive clones from a quail testis cDNA library which reveal the complete sequence for the quail protamine cDNA. The predicted amino acid sequence for the quail protamine contains the N-terminal tetrapeptide ARYR present in the N-terminal region of the mammalian protamines as well as several conserved motifs and arginine clusters. In addition the size of the quail protamine (56 amino acids) is closer to that of mammals (50 amino acids) than that of the chicken (61 amino acids). Altogether this data strongly suggests the existence of an avian-mammalian protamine gene line during evolution. Southern blot analysis suggests a small number of copies (2) per haploid genome (similar to that of chicken). The reported quail protamine cDNA sequence is the second avian protamine for which the amino acid sequence is available so far and provides new insights into vertebrate protamine function and evolution.

Amino Acid Sequence↗
PubMed1989

Chicken protamine genes are intronless. The complete genomic sequence and organization of the two loci.

A positive cosmid clone obtained from a pwe15-rooster DNA library using a chicken protamine cDNA probe reveals the complete sequence of the two loci for the rooster protamine genes. The organization of these two loci within the cosmid clone matches that of genomic DNA. The copy number per haploid genome is two. The sequence for the rooster protamine predicted from the coding region shows differences from that previously determined at the protein level (Nakano, M., Tobita, T., and Ando, T. (1976) Int. J. Peptide Protein Res. 8, 565-578). A recent re-determination of the rooster protamine amino acid sequence (28 residues from the N terminus) matches that predicted from the genome rather than the sequence of Nakano et al. (1976). Both loci are intronless and the gene is extremely GC-rich (88% in the coding region). The 5' region of the gene contains a typical TATAAA box, several CG boxes, as well as other characteristic motifs. The 3' region of the gene contains the polyadenylation signal and several GT repeats of known Z-DNA forming potential. A correlation between the functional map of the gene and the tendency of the DNA to bend or to adopt the Z-conformation is presented and possible roles for these conformations in the transcription of this gene are discussed.

Amino Acid Sequence↗
PubMed1988

Haploid expression of the rooster protamine mRNA in the postmeiotic stages of spermatogenesis.

cDNA clones were prepared from poly(A)+ mRNA isolated from a population enriched in postmeiotic rooster testes spermatogenic cells. A series of clones was sequenced at random and two partial sequences corresponding to the C-terminal coding and 3' untranslated region of the chicken protamine mRNA were obtained. The deduced amino acid sequence of this C-terminal coding region corresponds to the sequence previously described at the protein level for the chicken protamine, galline [Nakano, M., Tobita, T., and Ando, T. (1976), Int. J. Peptide Prot. Res. 8, 565-578]. To study the expression of this protamine gene, RNA was prepared from chicken testes at different stages of development, electrophoresed in formaldehyde-agarose gels, transferred to a nylon membrane, and hybridized with a rooster protamine cDNA probe. Two populations of mRNA of sizes ranging between 420 and 465 bases are expressed in postmeiotic rooster testis cells. To determine if there was a differential expression of the two populations of mRNA in the final postmeiotic haploid stages of spermatogenesis, RNA was purified from adult rooster cells separated at unit gravity according to their differences in size by the Staput technique. The RNA was similarly analyzed by Northern blots. The results indicate that round spermatids are enriched in the 465-nucleotide mRNA species, whereas in the final stage of elongated spermatids the 420-nucleotide species is the only one present, suggesting either post-transcriptional processing, the presence of two different sets of genes that are differentially expressed, or a single set of genes with differential promoter usage.

Amino Acid Sequence↗
PubMed1987

Factors affecting nucleosome disassembly by protamines in vitro. Histone hyperacetylation and chromatin structure, time dependence, and the size of the sperm nuclear proteins.

Histone displaced in vitro from nuclei by protamine competition display a higher degree of hyperacetylation than the residual histones. In addition, hyperacetylated core particle pools are disassembled in vitro with a higher efficiency than control or nonacetylated core particles and when analyzed by electron microscopy display an elongated shape (length/width ratio = 1.52 +/- 0.19) instead of the round compact shape of control nucleosomes (length/width ratio = 1.06 +/- 0.06). In the absence of histone hyperacetylation, the fish protamines, salmine and iridine (32-33 residues), are relatively inefficient in disassembling nucleosomal core particles in vitro as compared to the large (65-70 residues), tyrosine-containing protamines from rooster (galline), squid, and cuttlefish which disassemble nucleosomes in a range of protamine concentrations close to physiological. The fact that an artificially cross-linked salmine dimer acquires the ability of the large protamines from rooster, squid, and cuttlefish to disassemble core particles in vitro and also binds more tightly to the DNA, suggests that the size of the sperm nuclear protamines is a critical factor in this process. Even when the core histones of spermatid chromatin are hyperacetylated in the trout testis, the replacement process by iridine or salmine is slow and time-dependent in vitro. However, since spermiogenesis in trout occurs over several weeks, the slow in vitro nucleosome disassembly process by salmine is sufficient to allow complete displacement, thus supporting the hypothesis that a protamine-mediated displacement of the histones from DNA in vivo may take place in the salmonid fishes by a mechanism similar to that in the rooster, squid, and cuttlefish.

Acetylation↗
PubMed1987

Class I and II HLA antigen distribution in normal mucosa, adenoma and colon carcinoma: relation with malignancy and invasiveness.

HLA class I and II antigen expression was studied in normal mucosa, adenoma and colon carcinoma. Alkaline phosphatase anti-alkaline phosphatase (APAAP) staining techniques were used in cryostatic sections with anti-HLA-ABC and DR,DP,DQ monoclonal antibodies. All normal mucosa were intensely positive for HLA class I antigen expression, while failing to express class II molecules, except in mucosa adjacent to tumors (15/19 cases). All adenomatous polyps expressed HLA class I antigen, while the intensity of class II expression (DR greater than DQ greater than DP) was paralleled by the degree of dysplasia. In colon carcinoma, the loss of class I expression was seen in 4 out of 32 cases, and class II expression was found to be heterogeneous in 16 of these 32 cases (DR greater than DP greater than DQ). No relationship was noted between class II expression and degree of differentiation. However a correlation was seen between HLA-DR antigen expression and degree of invasiveness, mononuclear infiltrate and prognosis, according to Jass's criteria.

Adenocarcinoma↗
PubMed1986

Marked differences in the ability of distinct protamines to disassemble nucleosomal core particles in vitro.

In accordance with the results of classical experiments performed in vitro with calf thymus chromatin and the fish protamine salmine, we have observed that this highly basic, small molecular weight protamine cannot cause major displacement of histones from nucleosomal core particles at concentrations several times higher than physiological (arginine/nucleotide ratios 1-8) and that hyperacetylation of histones facilitates nucleosome disassembly. However, the avian protamine galline, with molecular weight and number of arginine residues almost twice those of common fish protamines, is able to displace the nucleosomal core histones from DNA in vitro at concentrations (arginine/nucleotide ratios 0.6-1.2) within the physiological range (0.8). Our results suggest that the binding of the avian protamine galline to chromatin could be directly involved in the rapid disassembly of nucleosomes that takes place during the nucleohistone nucleoprotamine transition in chicken spermiogenesis.

Animals↗
PubMed1982

Histone H4 hyperacetylation and rapid turnover of its acetyl groups in transcriptionally inactive rooster testis spermatids.

In order to study the relationship between acetylation of histones, chromatin structure and gene activity, the distribution and turnover of acetyl groups among nucleosomal core histones and the extent of histone H4 acetylation were examined in rooster testis cell nuclei at different stages of spermatogenesis. Histone H4 was the predominant acetylated histone in mature testes. Hyperacetylation of H4 and rapid turnover of its acetyl groups are not univocally correlated with transcriptional activity since they were detected in both genetically active testicular cells and genetically inactive elongated spermatids. During the transition from nucleohistone to nucleoprotamine in elongated spermatids the chromatin undergoes dramatic structural changes with exposition of binding sites on DNA (1). Hyperacetylation of H4 and rapid turnover of its acetyl groups could be correlated with the particular conformation of chromatin in elongated spermatids and might represent a necessary condition for binding of chromosomal proteins to DNA.

Acetates↗